ABC transporters are increasingly recognized as genes that play important roles in normal biology and in the therapeutic response to medications. Among the many mammalian ABC transporters that have been identified in the human genome, the functions of some have been deduced because "accidents of nature" produced mutations that render loss of function. However, for many ABC transporters the biological substrate is unknown and no adventitious accident of nature has been identified. One successful approach to identifying function is to develop drug resistant cell lines with resistance due to drug efflux and then identify an ABC transporter that dominantly imparts this resistance by drug efflux. This was the case for the ABC transporter Mrp4 that was an orphan ABC transporter until our studies first determined that it was unique in its ability to transport nucleotide analogs used in HIV therapy. During the last funding period of this application we used cell line model systems to expand Mrp4's biological substrates (e.g., cAMP and conjugated steroids) and the drugs (mercaptopurine nucleotides) it confers resistance. We also discovered that Mrp4 was expressed in the liver and it's expression dramatically increased as an adaptive response to the accumulation of cholestatic bile acids. Our current research will focus on understanding the function of Mrp4 in vivo. I have developed an Mrp4 nullizygous mouse with T cells that have impaired cAMP efflux. In contrast, T cells from Mrp4 +/+ mice readily efflux cAMP. Moreover, these Mrp4 -/- T cells have a defect in proliferation. The relationship between Mrp4 regulation by the second messenger cAMP and the relation with proliferation will be further explored in Aim 1 of the current application. Because our results show that Mrp4 has a role in the transport of cholestatic conjugated bile salts and is upregulated under cholestatic conditions, I will determine if Mrp4 absence renders the liver more susceptible to cholestatic injury. I will also use the Mrp4 -/- animals to identify mechanisms (Aim II) in the liver that compensate under cholestasis, but are independent of Mrp4. I will also determine if nuclear receptors (e.g., the pregnane X receptor, PXR) contribute to Mrp4 upregulation in Aim II by using nuclear receptor knockout models. Finally, to more fully elucidate the signaling processes and cross talk among these Mrp4 activation pathways we will develop an Mrp4-GFP knock-in mouse model in Aim III. Cumulatively, these studies will increase our knowledge of this increasingly important ABC transporter and provide insight into how Mrp4 plays a role in hematopoietic cells and the liver [unreadable] [unreadable]